PROJECT SUMMARY Genomic DNA is constantly damaged by endogenous or exogenous agents or processes. Cells have evolved several DNA repair pathways including DNA base excision repair (BER) to fix the damage. BER repairs the vast majority of single base lesions, including a major oxidative DNA lesion 8-oxoguanine (8-oxoG). 8-oxoG is highly mutagenic due to its ability to form both a Watson- Crick base pair with correct dCTP and a Hoogsteen base pair with incorrect dATP during translesion DNA synthesis (TLS), which can lead to cancer. To repair 8-oxoG, human cells use four enzymes to carry out sequential BER reactions and they are human 8-oxoG DNA glycosylase (hOGG1), AP endonuclease (APE1), DNA polymerase ? (hPol?), and DNA ligase III/XRCC1. Although these enzymes have been investigated for years, there are many unresolved or controversial mechanistic questions about their catalytic functions. For example, it is not completely clear how hPol?, which possesses a DNA polymerase activity and a 5?-deoxyribose- 5-phosphate lyase (dRPase) activity, executes both gap-filling DNA synthesis and removal of 5?- dRP during BER. Moreover, the role of a newly discovered third divalent metal ion in hPol?- catalyzed nucleotide incorporation remains unclear. Additionally, the mechanisms of hOGG1- catalyzed base excision and strand scission remain controversial. Finally, it has been proposed that there must be some mechanism of coordination between BER proteins to allow efficient and rapid repair of DNA damages. The long-term objectives of the Principle Investigator (PI) are to use biochemical and biophysical methods to elucidate the detailed kinetic and structural mechanisms for the BER enzymes and dynamic protein/protein and protein/DNA interactions in this important DNA repair pathway. In Aim 1 of this proposal, the PI will employ conventional and time-dependent crystallography to establish the structural basis for the dRPase activity of hPol?, define the role of the third divalent metal ion, watch the lesion bypass and extension steps of TLS across 8-oxoG by hPol?, and refine the catalytic mechanism of hOGG1. In Aim 2, the PI will use single-molecule Frster resonance energy transfer (FRET) and colocalization single-molecule spectroscopy (CoSMoS) to characterize the interaction between hOGG1 and APE1 and to elucidate the mechanism of BER coordination. Completion of this proposal will better define the catalytic roles of critical active site residues in the hPol? dRPase domain and hOGG1, provide new information for the role of the third divalent metal ion in the DNA polymerase-catalyzed reaction, and give unprecedented evidence for the modes of coordination during BER.